Quantitative Detection of Fusarium Species in Wheat Using TaqMan

Fusarium head blight (FHB) of wheat and other small-grain cereals is a disease complex caused by several fungal species. To monitor and quantify the major species in the FHB complex during the growing season, real-time PCR was developed. TaqMan primers and probes were designed that showed high specificity for Fusarium avenaceum, F. culmorum, F. graminearum, F. poae and Microdochium nivale var. majus. Inclusion of an internal PCR control and serial dilutions of pure genomic DNAs allowed accurate determination of the concentration of fungal DNA for each of these species in leaves, ears as well as harvested grains of winter wheat. The DNA concentration of F. graminearum in grain samples correlated (r ²= 0.7917) with the incidence of this species on the grain as determined by isolation from individual kernels. Application of the TaqMan technology to field samples collected in 40 wheat crops in the Netherlands during the growing season of 2001 revealed that M. nivale var. majus predominated on leaves early in the season (GS 45-65). Ears and harvested grains from the same fields, however, showed F. graminearum as the major species. In 2002, grain samples from 40 Dutch fields showed a much wider range of species, whereas in ears from 29 wheat crops in France, F. graminearum was the predominant species. The concentration of DON correlated equally well with the incidence of the DON-producing species F. culmorum and F. graminearum in the grain samples (r ²= 0.8232) as well as with total DNA of both these species (r ²= 0.8259). The Fusarium TaqMan technology is an important tool to quantify and monitor the dynamics of individual species of the complex causing FHB in cereals during the growing season. This versatile tool has been applied in a comparison of different genotypes, but can also be applied to other disease management systems, e.g. fungicide treatments.     

Modified bases characterized in intact DNA by mass-analyzed ion kinetic energy spectrometry

Pyrolysis of DNA into a chemical ionization source yields protonated bases and other base-containing ions. Kinetic energy spectra allow the characterization of the bases 5-methylcytosine and 1-methyladenine from underivatized salmon sperm DNA. Isomeric bases are distinguishable with this technique.     

Ovine Johne's disease: the risk associated with sheep imported into Western Australia

Objective To assess the risk of Johne's disease not being detected in sheep imported from New South Wales into Western Australia.
Design A stochastic simulation model.
Procedure The process of importing sheep was broken down into steps and numbers or probabilities assigned to each. Controls on the movement of sheep included surveillance tests in source flocks and serological tests on sheep in consignments before and after transportation to Western Australia. The model calculated the risk of occurrence of Johne's disease in Western Australia and the success of the agar gel immunodiffusion test in identifying consignments with infected sheep.
Results Negative surveillance tests in source flocks reduced the risk to about one twentieth of that when no surveillance tests were required. On average, Johne's disease was predicted to be introduced once in every 3 to 7 years when no testing of either the source flock or the sheep in consignments was required. When negative surveillance tests only were required the interval increased to once in every 63 to 111 years and, with the additional requirement that all sheep in each consignment must have a negative test before and after transport, the interval further increased to once in every 125 to 333 years. When only sheep in consignments were tested, the interval was calculated to be 8 to 14 years.
Conclusion A requirement that imports be derived from flocks which had negative surveillance tests to Johne's disease would provide significantly greater protection for the sheep industry in Western Australia.     

Use of Vaginal Electrical Resistance to Diagnose Oestrus,Dioestrus and Early Pregnancy in Synchronized Tropically Adapted Beef Heifers

The primary objective of this study was to determine whether a single measurement of intravaginal electrical resistance (VER), using the commercially available Ovatec^® probe, can discriminate between dioestrus and oestrus in Bos indicus females, which had been treated to synchronize oestrus. Santa Gertrudis heifers (n = 226) received one of three oestrous synchronization treatments: double PGF_2α 10 days apart, 8‐day controlled internal drug release (CIDR) treatment or CIDR pre‐synchronization + PGF_2α 10 days after CIDR removal. The heifers were inseminated within 12 h following observed oestrus, or, if not observed, at a fixed time approximately 80 h, following the last synchronization treatment. They were palpated per rectum for signs of pregnancy 9 weeks after artificial insemination (AI). Vaginal electrical resistance measurements were taken at the completion of synchronization treatments (presumed dioestrus), immediately prior to AI (oestrus), and then at 3 and 9 weeks post‐AI. Mean VER differed between presumed dioestrus and oestrus (113.7 vs 87.4, p < 0.001). The area under the receiver operating characteristics (ROC) curve was 0.925, indicating that VER was highly discriminatory between dioestrus and oestrus. Vaginal electrical resistance at time of AI was negatively associated with odds of conception when all inseminations were included in the analyses [odds ratio (OR) = 0.97; 95% CI 0.95–1.00; p = 0.018], but not when fixed time AIs were excluded (OR = 1.00; 95% CI 0.97–1.03; p = 0.982). Mean VER readings differed between pregnant and non‐pregnant animals at both 3 weeks (120.5 vs 96.7, p < 0.001) and 9 weeks (124.0 vs 100.3, p < 0.001) post‐AI. However, 3‐ and 9‐week VER measurements were not highly discriminatory between pregnancy and non‐pregnancy (area under ROC curve = 0.791 and 0.736, respectively). Mean VER at time of AI for animals diagnosed in oestrus differed between each of the oestrous synchronization treatments (84.7, 73.6 and 78.9, groups 1–3 respectively, p < 0.001). These findings suggest that measurement of VER may improve accuracy of oestrus diagnoses when selecting cattle for AI following oestrous synchronization programmes involving tropically adapted cattle.     

Myofibroblastic fibrosarcoma with multifocal osseous metaplasia at the site of equine influenza vaccination

We describe a fibrosarcoma in a 12-year-old Quarterhorse × Arabian gelding as a sequela to equine influenza vaccination. Shortly after the second vaccination, swelling at the site was noticed by the owner and it continued to increase in size over the following 6 months. Biopsy of the mass indicated a fibrosarcoma had developed at the vaccination site. It was approximately 20 cm in diameter and elevated well above the level of the skin. There was no clinical evidence of metastases to the lungs or local lymph nodes. Surgical resection of the mass was performed and the wound healed by first and second intention. Histopathological examination and immunohistochemical staining confirmed a myofibroblastic fibrosarcoma with multifocal osseous metaplasia. To the authors' knowledge, this is the first equine case of a vaccine-associated fibrosarcoma.     

The incidence, cost and factors associated with foot lameness in dairy cattle in south-western Victoria

A survey of 73 dairy farms in south-western Victoria was conducted to assess the cost and mean herd incidence of foot lameness for the period from calving to the end of November, 1985, and to identify the herd, management and environmental factors associated with foot lameness. The mean herd size was 125 cows (range 82 to 220). Lameness occurred in 64 (88%) herds, and the mean herd incidence was 7.0% (range 0.0 to 30.9%). The main clinical signs associated with lameness were the presence of overworn and/or bruised soles, or stones lodged in the interdigital cleft. Factors associated with lameness were: property and herd size, age of cow, bail feeding, voluntary entry into the bails, and features of the farm track including its length, the presence of steep slopes, the type of surface material, presence and treatment of broken sections and maintenance including rolling history. The association of these factors with specific clinical signs was examined. The mean cost was estimated to be $42.90 per lame cow due to loss of production, treatment, the culling or death of lame cows, and extra man hours spent managing lame cows. It was concluded that the site, construction, maintenance and use of the farm track were of major importance to the incidence of lameness in herds in this area and recommendations for reducing lameness are made.     

A case study of seed exchange networks and gene flow for barley (Hordeum vulgare subsp. vulgare) in Morocco

Local patterns of seed regeneration and trade that occur outside the formal breeding sector (seed exchange networks) can have a strong influence on the genetic diversity and evolution of traditional crop varieties. Despite this, little is known about the extent to which seed exchange networks influence gene flow and genetic structure in traditional crop varieties. Here we study barley (Hordeum vulgare subsp. vulgare) in rural communes of Northern Morocco in 2008 and 2009. We quantified seed regeneration and exchange by farmers within the seed exchange network using structured interviews. Using SSR markers, we also quantified the neutral genetic diversity and structure of a complex of traditional varieties referred to as Beldi that is managed in this exchange network. The majority of farmers (>88 %) report cultivating Beldi. Most seeds of Beldi (70–90 %) are maintained on-farm, while the remainder of seeds are obtained from local markets within the commune. Beldi has high genetic diversity and there is weak but significant genetic structure between communes (F_ST = 0.031). From SSR marker data there is evidence of a high level of gene flow between communes not reported in interviews. Seeds purchased in local markets likely represent seeds from a larger geographic region, leading to lower genetic structure among communes than expected based on the reported level of on-farm seed regeneration and local sourcing of seed. We discuss the implications of this seed exchange network for the conservation of traditional barley varieties in the study region.     

Mutation rates and dominance levels of genes affecting total fitness in two angiosperm species

Theories about the evolution of sex and the effects of inbreeding depend on knowledge of the mutation rate and dominance level of deleterious alleles affecting total fitness. In two species of largely self-fertilizing annual plants, minimal estimates of such mutation rates were found to be 0.24 to 0.87 per sporophyte genome per generation, but confidence intervals exceeded 1.0 in each of the four populations. Dominance levels were near zero in one species and intermediate (0.28 to 0.35) in the other. These results suggest that the detrimental effects of inbreeding are a result of new partially recessive mutations rather than overdominance.